Activation of skeletal muscle fibers underlies all bodily movement, and is initiated by electrical depolarization of their transverse tubules (TTs), which penetrate the fibers at each sarcomere. Membrane voltage sensors in the TT dihydropyridine receptor (DHPR) respond to TT depolarization, and trigger Ca2+ release via the abutting ryanodine receptor (RyR)/Ca2+ release channels in the adjacent sarcoplasmic reticulum membrane. In frog muscle, the "macroscopic" Ca2+ release caused by fiber depolarization has been shown by us and others to be composed of discrete local events, the Ca2+ sparks. In Aim 1 we will use ultra high speed confocal imaging to characterize the gating properties of the few channels that underlie each spark, and to determine whether the group of channels always gates in unison, or may sometimes gate independently during a spark. In Aim 2 we will examine Ca2+ sparks in mammalian muscle, where they do not seem to be the basic unit of physiological Ca2+ release, but instead appear under abnormal or pathological conditions and during muscle repair, possibly due to DHPR / RyR uncoupling. We will study the properties and possible functional roles of Ca2+ sparks in mammalian muscle using adult muscle fiber dedifferrentiation in culture as a model system for myoblast fusion and fiber remodeling, which is important for muscle repair in disease, in dystrophic (mdx) muscle and in muscle from aging mice. In Aim 3 we will investigate the modulation of macroscopic Ca2+ release in mammalian muscle by endogenous protein ligands and by experimental RyR "domain" peptides. We will use high speed (<50 us/line) line- or band-scan confocal imaging of fibers containing Ca2+ indicators to monitor Ca2+ release during depolarization, together with acute or chronic application of proteins, protein fragments or chimeric constructs, including fluorescent tags for sarcomeric localization, or of small peptides that may disrupt the normal endogenous coupling process. Among the molecules to be investigated for participation in, or modulation of the physiologic (ie, voltage activated) release process will be the DHPR beta subunit, the Ca2+ binding protein S100A1 and "domain" peptides of the RyR. This project will elucidate basic molecular mechanisms regulating Ca2+ release in skeletal muscle and the roles of Ca2+ sparks in muscle disease, damage and repair, and will provide a better understanding of abnormal regulation of Ca2+ release channels in muscle disease.